NF-κB Activation and Severity of Gastritis in Helicobacter pylori ...

NF-jB Activation and Severity of Gastritis in Helicobacterpylori-Infected Children and AdultsPatrick Bontems,*,† Ezra Aksoy,†,‡ Alain Burette,§ Val�erie Segers,¶ Carine Deprez,¶ Franc�oise Mascart†,**and Samy Cadranel*

*Paediatric Gastroenterology-Hepatology, Queen Fabiola Children’s University Hospital, Universit�e Libre de Bruxelles, Av JJ Crocq 15, 1020

Brussels, Belgium, †Laboratory of Vaccinology and Mucosal Immunity, Universit�e Libre de Bruxelles, Route de Lennik 808, 1070 Brussels, Belgium,‡Centre for Cell Signalling, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK, §Department of

Gastroenterology, Nouvelle Clinique de la Basilique, Rue Pangaert 37, 1083 Brussels, Belgium, ¶Department of Pathology, University Hospital

Brugmann, Universit�e Libre de Bruxelles, Place Van Gehuchten 4, 1020 Brussels, Belgium, **Immunobiology Clinic, Academic Hospital Erasme,

Universit�e Libre de Bruxelles, Route de Lennik 808, Brussels, Belgium

Keywords

Helicobacter pylori, NF-jB activation,

gastroduodenal ulcer, children, gastritis.

Reprint requests to: Patrick Bontems, Paediatric

Gastroenterology-Hepatology, Queen Fabiola

Children’s University Hospital, Universit�e Libre de

Bruxelles, Av JJ Crocq 15, 1020 Brussels,

Belgium. E-mail: [email protected]

Abstract

Background: In contrast to adults, Helicobacter pylori gastritis in children is

reported as milder and ulcer disease as uncommon, but unequivocal data

are lacking.

Objectives: To compare the frequency of gastro-duodenal ulcers in children

and adults as well as the proportion of Helicobacter pylori infection in these

patients and to study the effect of chronological age on NF-jB activation

and on severity of gastritis.

Design: Patients referred in one pediatric and one adult facility for upper GI

endoscopy were included. Gastric biopsies were obtained in consecutive Heli-

cobacter pylori-infected patients and age-matched negative controls for immu-

nohistochemistry and electrophoresis mobility shift assay. Three age groups

were defined: younger than 8 years, 8–17 years, and adults.

Results: Peptic ulcer disease was less frequent in children and less fre-

quently associated with Helicobacter pylori infection. When comparing

infected subjects to controls, densities of neutrophils and CD20 cells in the

lamina propria increased in all age groups, CD3 cells increasing only in

patients older than 8 years and CD8 cells only in adults. NF-jB-p65-positivecells were also increased only in infected adults as well as NF-jB-bindingactivity. A positive correlation was found between age and densities of neu-

trophils and CD3, but not of CD8 or CD20 cells.

Conclusion: Peptic ulcer disease was less frequent in children and less fre-

quently caused by Helicobacter pylori infection. The different clinical outcome

of the infection in children can be the consequence of the lower mucosal

immune response.

Helicobacter pylori (H. pylori) is a gram-negative microor-

ganism that colonizes the mucus coat overlying the gastric

epithelium and causes chronic/active gastritis, peptic ulcer

disease (PUD), gastric mucosal atrophy, intestinal metapla-

sia, gastric adenocarcinoma, and gastric lymphoma [1].

Although the prevalence of infection varies geographically

and is declining over time in developed countries, the

organism still infects approximately one half of the world’s

population [2]. Progression from gastritis toward more

severe disease states appears to be influenced by both bac-

terial determinants and host immune response [3,4].

Immune response against H. pylori involves innate

components represented mainly by neutrophils and

adaptive immunity involving systemic and mucosal

H. pylori-specific antibody production as well as a com-

plex mix of Th1, Th17, and Treg immune responses

[5–9]. Interactions of H. pylori strains and bacterial

compounds with diverse host epithelial and cytoplasmic

signaling pathways such as Toll-like receptors (TLRs) or

nucleotide-binding oligomerization domain (NLRs) is

followed by the activation of NF-jB, a transcription

factor regulating activation of genes responding to

© 2014 John Wiley & Sons Ltd, Helicobacter 19: 157–167 157

Helicobacter ISSN 1523-5378

doi: 10.1111/hel.12118

immune or inflammatory signals [8–16]. These immune

responses fail to eradicate H. pylori infection and cer-

tainly contribute to disease pathogenesis, in part due to

tissue remodeling trough inflammation and activation

of matrix metalloproteinase [17–22]. Although NF-jBhas a key role in mediating gastric mucosal inflamma-

tion caused by H. pylori, its activation has not been

studied yet in infected children.

The systemic humoral response to H. pylori infec-

tion is weaker and variable in children [23,24]. In

addition, a lower IFN-c secretion in the stomach and a

lower infiltration by IFN-c-secreting cells characterize

the mucosal immune response to H. pylori in children

compared with infected adults [6,7]. Moreover, regula-

tory T-cell response to H. pylori infection also predomi-

nates in children instead of Th17 cell [25–27]. These

evidences suggest that a weaker immune response

could protect the child from more severe gastroduode-

nal damages due to the infection. A lesser inflamma-

tory cell mucosal infiltration has been reported in

children, but there are still controversies especially

concerning the lack or reduced infiltration of the gas-

tric mucosa by polymorphonuclear cells in H. pylori-

infected children [28]. Indeed, most studies are not

really comparative because adult patients were not

included (the reference to adult patients is only histor-

ical). Moreover, three comparative studies that used a

semiquantitative scoring of gastritis (the Update Syd-

ney system [29]) give conflicting results [25,30,31].

Using the same score in a pilot study, we also failed to

demonstrate a difference in the severity of gastritis

between children and adults (P. Bontems, A. Neuman-

Ova, P. Heimann, J.M. Devaster, V. Segers, C. Deprez,

S. Cadranel, unpublished observations).

In the present study, we compared the frequency of

gastric and duodenal ulcer disease between children

and adults and the proportion of lesions related to

H. pylori infection as well as the cellular density of

immune cells in the gastric mucosa using immunohisto-

chemical staining and a morphometric method and the

activation of NF-jB by the same method and by

electrophoresis mobility shift assay (EMSA).

Patients and Methods

Frequency of Gastric and Duodenal Ulcers

The source of data was two different endoscopy facili-

ties, one for children and one for adults, located very

closely in Brussels. In our routine practice, gastric biop-

sies for histologic analysis and microbiologic culture

were systematically taken from children and adults

whenever not contraindicated. All patients undergoing

an upper GI endoscopy between January 2007 and

December 2008 were enrolled to compare the frequency

of gastric and duodenal ulcers and the proportion of

ulcers associated with H. pylori infection. Only ulcers

with a diameter of 5 mm or larger were considered. The

following items were recorded: the presence of gastric or

duodenal ulcers, H. pylori status, age, and gender.

At least two biopsies were taken from the antrum

and two more from the fundus for histology and for

microbiologic culture. The biopsies were processed

according to the previously described methods [32].

Briefly, sections from formalin-fixed, paraffin-embed-

ded biopsies were stained with hematoxylin/eosin to

detect histologic features of inflammation (in accor-

dance with the Updated Sydney system [29]) and with

Giemsa to detect H. pylori-like microorganisms. Biopsy

specimens for culture were dipped with sterile cotton in

a semisolid agar transport medium (Cultiplast, LP Itali-

ana SPA, Milan, Italy). Less than 4 hours later, the

samples were grounded at 10000 rpm for 15 second

with an electric homogenizer (Ultraturrax, Staufen,

Germany) and then inoculated onto selective Columbia

blood agar and incubated under microaerophilic (5%

O2 – 10% CO2 – 85% N2) conditions at 37 °C for up to

7 days. H. pylori infection was confirmed when culture

and histology were both positives. Discrepancies

between histology and culture were resolved using an

additional diagnostic test (urease test or 13C-urea breath

test). H. pylori culture in our departments was concor-

dant with histology in 98% [32].

Cellular Density of Immune Cells in the Antral

Mucosa

Another database was constructed with all the patients

undergoing an upper GI endoscopy during 1 year except

those presenting with erosive esophagitis, gastric, or duo-

denal ulcers. Those with inflammatory bowel disease,

celiac disease, immunologic disorders as well as those

treated during the preceding 4 weeks with antibiotics,

proton-pump inhibitors, nonsteroidal anti-inflammatory

drugs, steroids, or under immunosuppressive drugs were

also excluded. From this database, 30 consecutive

H. pylori-infected patients and 30 negative controls were

selected in the proportion of 10 in each age group

(<8 years, 8–17 years and adults). Age groups were arbi-

trarily defined because the humoral response was found

weaker in children younger than 8 years, whereas it was

comparable with adults in older children [23].

Polymorphonuclear cells were counted on hematoxy-

lin- or eosin-stained sections and neutrophils differenti-

ated using morphologic criteria. B cells (anti-CD20),

T cells (anti-CD3), and T cells subsets (anti-CD8) were

© 2014 John Wiley & Sons Ltd, Helicobacter 19: 157–167158

NF-jB and severity of H. pylori gastritis Bontems et al.

counted on sections using an indirect immunoperoxidase

technique. Formalin-fixated tissue sections (causing

inter- and intramolecular cross-linking masking

antigenic epitopes) from the antrum were submitted to

microwave oven treatment. Deparaffinized and rehy-

drated tissue sections, placed in a 0.01 mol/L citrate buf-

fer at pH 6, were heated twice during 5 minute and then

rinsed in Tris-buffered saline at pH 7.4. Sequential

sections were then pre-incubated with rabbit antiserum

to reduce nonspecific staining and with 0.2% H2O2 to

inhibit endogenous peroxidases. Optimal dilution of the

primary antibody layer was added and incubated for

1 hour at room temperature, followed by optimal

dilution of the secondary (biotinylated) antibody layer,

the streptavidin-biotin complex, and the 3,30-Diam-

inobenzidine tetrahydrochloride (DAB) which develops

the peroxidase. The sections were counterstained with

hematoxylin. Monoclonal mouse anti-human antibodies

were used as primary antibody and biotinylated rabbit

anti-mouse antibody as secondary antibody (Dako Ltd,

Glostrup, Denmark). In place of the primary antibody, a

nonimmune serum from the same species was used as

negative control staining, whereas human tonsil sections

were used for positive control staining.

Cellular densities were counted at 4009 magnifica-

tion in the lamina propria using a calibrated eyepiece

graticule (Leitz, Wetzlar, Germany) defining a limited

area (four different areas on two different sections) and

in the epithelium on a 500 epithelial cells surface.

Slides were counted by two independent observers.

Activation of the Transcription Factor NF-jB in

the Antral Mucosa

The number of cells expressing activated NF-jB was

determined using the same indirect immunoperoxidase

technique described above using an anti-a-p65 subunit

antibody (Biognost, Heule, Belgium) that recognizes only

the activated form of NF-jB. The densities of cells express-ing the activated form of NF-jBwere then determined.

Additionally, DNA-binding activity of NF-jB was

determined in protein extracts obtained from consecu-

tive patients with nonulcer dyspepsia (six consecutive

children: three of them being infected by H. pylori and

the three others serving as controls; eight consecutive

adults: three infected by H. pylori and five controls) by

EMSA. Biopsies were immediately snap-frozen in liquid

nitrogen after sampling and stored at �70 °C until

assayed. To prepare total protein extracts, biopsies were

suspended in 400 lL of buffer 10 mmol/L HEPES (pH

7.8), 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.5 mmol/L

DTT, 19 protease inhibitor cocktail (Sigma-Aldrich,

Bornem, Belgium), 0.2 mmol/L PMSF (Roche Diagnos-

tics, Basel, Switzerland), and 0.5% Triton X-100

(Sigma-Aldrich) for 10 minute on ice after mechanical

homogenization. Suspensions were then centrifuged at

4 °C for 5 minute, and the cytoplasmic proteins in the

supernatants carefully removed. Protein concentrations

were determined by BioRad Protein Assay Reagent

(Bio-Rad Labs., Hercules, CA, USA). The double-

stranded binding oligonucleotides for consensus NF-jB50 – AGT TGA GGG GAC TTT CCC AGG C – 30 were

obtained from Santa CruzBiotechnology. 50 – AGT TGA

GGG GAC TTT CCC AGG C – 30-NF-jB mutant oligos

were created with a “G’’?”C’’ substitution (Santa Cruz

Biotechnology, Dallas, TX, USA). Oligonucleotides were

end-labeled with [c-32P] ATP (Amersham, Little

Chalfont, UK) using T4 polynucleotide kinase (Roche

Diagnostics). For the binding reaction, 30 lg of each of

the extract was added to a reaction mixture containing

2 lg poly(dI-dC) (Pharmacia, San Diego, CA, USA),

4 lL of 59 binding buffer (10 mmol/L HEPES, (pH

7.8), 50 mmol/L KCl, 1 mmol/L EDTA, 5 mmol/L

MgCl2, 10% glycerol), and 30,000 cpm of [32P]-labeled

oligonucleotide in a final volume of 20 lL and was

incubated at room temperature for 15 minute. The free

and protein-bound oligonucleotides were resolved by

electrophoresis on a 5% polyacrylamide gel in a 0.59

Tris-borate EDTA buffer. After electrophoresis, the gel

was dried and exposed to autoradiography film (East-

man Kodak Co., Rochester, NY, USA). The images were

processed with Adobe Photoshop CS3 extended (Adobe

Systems Inc, San Jos�e, CA, USA) and the densitometric

analyses of the NF-jB-binding bands were analyzed

using ImageJ software. (Rasband WS, National Insti-

tutes of Health, Bethesda, MD, USA).

CagA Strains

The cagA gene encodes a cytotoxin-associated gene A

antigen (CagA) protein, an important bacterial viru-

lence factor. Genomic DNA from the bacterial strains

was PCR-amplified using two sets of synthetic oligonu-

cleotide primers as described elsewhere [33]. The

amplified PCR products were resolved in 1% agarose

gels containing Tris/borate/EDTA using 100 bp (Gibco

BRL; Gaithersburg, MD, USA) as a molecular weight

marker. The agarose gels were stained with ethidium

bromide and viewed under short-wavelength UV light.

The strains were considered to be CagA positive when

at least one of the reactions was positive.

Ethics

This study was approved by the Research Ethical Com-

mittee of the Medical Faculty of the Universit�e Libre de


Bontems et al. NF-jB and severity of H. pylori gastritis

Bruxelles, and informed consent was obtained from the

patients or their parents.

Data Analysis

The statistical analysis was performed using Statistical

Package for Social Sciences (SPSS; IBM, Armonk, NY,

USA), version 21. The medians and the ranges were

calculated for all continuous variables as well as reparti-

tion for qualitative parameters. The performance of the

Pearson chi-square test was calculated for unpaired

qualitative variables. The distributions of non-Gaussian

continuous variable between two groups were com-

pared using the nonparametric Mann–Whitney U-test

and the Kruskal–Wallis test for more than two groups.

Spearman’s rank correlation was used to measure the

statistical dependence between two continuous vari-

ables. Statistical significance was set up at the p < .05

level, and all p values were two-tailed.

Results

Frequency of Gastric and Duodenal Ulcers

Through 2007–2008, 1484 upper GI endoscopies were

performed in 1279 pediatric patients and 1046 endos-

copies in 1010 adult patients. Gastric or duodenal ulcers

with a diameter of at least 5 mm were found less fre-

quently in children than in adults (20/1279 children –

1.6% vs 58/1010 adults – 5.7%, OR 0.30, 95% CI

0.10–0.86, p = .02). Ulcers were also less frequently

associated with H. pylori infection in children than in

adults (8/20 vs 40/58, OR 0.26, 95% CI 0.16–0.78,

p < .0001). The median age for children with ulcer was

14.5 years (range 1.7–16.1) and 7.0 years (range 0.1–

17.9) for those without ulcer.

Cellular Density of Immune Cells in the Antral

Mucosa

Neutrophils

Neutrophil densities were compared in the antrum and

in the fundus (Fig. 1) in the different age groups.

Among the controls, there was virtually no neutrophil

within the lamina propria and the epithelium of the

antral and fundic mucosa. Among the H. pylori-infected

patients, the median neutrophil count per field

increased significantly in all age groups in the lamina

propria of the antrum and the fundus. In the epithe-

lium, the neutrophil count increased only in adults in

the antrum. When considering the effect of age, we

found a significant correlation with neutrophil density

in the antrum of infected patients (Spearman’s rank

correlation: lamina propria r = .456, p = .003 – epithe-

lium r = .500, p = .001), but not in the fundus of these

patients or in uninfected controls.

CD20, CD3, and CD8 cells

Among the controls (Fig. 2), there was virtually no

CD20-positive cell in the lamina propria and in the epi-

thelium except a very low density found in the lamina

propria of adults.

Among the H. pylori-infected patients, there was a

significant increase in the densities of CD20-positive

cells in all age groups in the lamina propria, but not in

the epithelium. When considering the effect of age, no

correlation found with CD20 density in the lamina pro-

pria or in the epithelium of infected patients or in

uninfected controls.

Among the controls, CD3-positive cells were found

in the lamina propria and in the epithelium. The den-

sity was slightly higher in the lamina propria of the

younger children compared with older ones and to

adults, whereas in the epithelium, the densities were

comparable in all age groups.

Among the H. pylori-infected patients, the CD3

densities in the lamina propria and in the epithelium

remained comparable with controls in children younger

than 8 years and increased significantly in older chil-

dren and in adults. When considering the effect of age,

a positive correlation was found with CD3 density in

the lamina propria of infected patients (r = .516,

p = .001), but not in the epithelium or in uninfected

controls.

Among the controls, there were some CD8-positive

cells in the lamina propria and in the epithelium, the

densities being lower than those of CD3 cells. There

was no difference between age groups.

Among the H. pylori-infected patients, we only

observed an increased density of CD8 cells in the lam-

ina propria of adult patients. However, there was no

correlation between age and density of CD8 cells in the

lamina propria or in the epithelium of infected patients

or uninfected controls.

Activation of the Transcription Factor NF-jB

Density of cells expressing activated NF-jB

The number of cells expressing activated NF-jB in the

lamina propria and in the epithelium of the antral

mucosa was increased in infected adults, whereas it is

similar to controls in infected children (Fig. 3). The lack

of activation of NF-jB correlates with the lower



recruitment of neutrophils in children compared with

adults (r = .672, p < 10�4).

Electrophoresis Mobility Shift Assay

DNA-binding activity of NF-jB is similar in infected

children and in controls, but largely increased in

infected adults (Fig. 4).

Helicobacter pylori Density and CagA-Positive

Strains

Helicobacter pylori density

The densities of H. pylori in the antral and the fundic

mucosa were evaluated using the Sydney system, a

semiquantitative score. Although there is a tendency to

have a higher bacterial load between the pediatric age

groups, the differences were not significant (Fig. 5).

Moreover, the severity of the recruitment of immune

cells in the mucosa was not correlated with the bacterial

load.

Frequency of CagA-positive strains

Infection with a CagA-positive strain was found in 5 of

10 children younger than 8 years, 4 of 10 in the 8–17

years age group, and in 5 of 10 adults (NS). Densities of

immune cell in patients with a CagA-positive strain were

generally slightly higher in each age group, but a statisti-

cal significance was only observed for the number of

neutrophils in the epithelium of adults (p = .02).

Figure 1 Comparison of the densities of neutrophils in the lamina propria and in the epithelium of uninfected and H. pylori-infected children

(below 8 years of age and 8–17 years) and adults with nonulcer dyspepsia. There are 10 patients in each group. Neutrophil densities are

expressed as number of cells per surface unit, defined using a calibrated eyepiece graticule at 4009 magnification, in the lamina propria and as

number of cells per 100 epithelial cells surface in the epithelium. Data are compared between groups using the Mann–Whitney U-test (NS, not

significant). In this boxplot representation, ends of the whiskers represent �1.5 interquartile range; outliers are indicated by a circle and extreme

outliers (outside �3 interquartile range) by a star.



Figure 2 Comparison of the densities of CD20-stained cells (B cells), CD3-stained cells (T cells), and CD8-stained cells (cytotoxic T cells) cells in

the lamina propria and in the epithelium of uninfected and H. pylori-infected children (below 8 years of age and 8–17 years) and adults with non-

ulcer dyspepsia. There are 10 patients in each group. Neutrophil densities are expressed as number of cells per surface unit, defined using a cali-

brated eyepiece graticule at 4009 magnification, in the lamina propria and as number of cells per 100 epithelial cells surface in the epithelium.

Data are compared between groups using the Mann–Whitney U-test (NS, not significant). In this boxplot representation, ends of the whiskers rep-

resent �1.5 interquartile range; outliers are indicated by a circle and extreme outliers (outside �3 interquartile range) by a star.



Figure 3 Comparison of the densities of cells stained using an anti-p65 NF-jB antibody that binds to the active form of NF-jB in the lamina pro-

pria and in the epithelium of uninfected and H. pylori-infected children (below 8 years of age and 8–17 years) and adults with nonulcer dyspepsia.

There are 10 patients in each group. Neutrophil densities are expressed as number of cells per surface unit, defined using a calibrated eyepiece

graticule at 4009 magnification, in the lamina propria and as number of cells per 100 epithelial cells surface in the epithelium. Data are compared

between groups using the Mann–Whitney U-test (NS, not significant). In this boxplot representation, ends of the whiskers represent �1.5 inter-

quartile range; outliers are indicated by a circle and extreme outliers (outside �3 interquartile range) by a star.

A

B

Figure 4 Cell extracts were prepared from antral biopsies of consec-

utive patients with nonulcer dyspepsia. The interaction between NF-

jB and consensus radioactively labeled DNA probes was detected by

gel electrophoresis (Eight consecutive adults, five uninfected controls,

nos. 1–4 and three adults infected with H. pylori nos 5–7 followed by

six children; three uninfected controls nos. 8–10 and three infected

with H. pylori nos 11–13). The double arrows indicate NF-jB-binding

activity while single arrow represents nonspecific binding (ns). Pr,

probe alone.

A

B

Figure 5 Comparison of the H. pylori densities in the antrum (A) and

in the fundus (B) between the infected children (below 8 years of age

and 8–17 years) and the adults with nonulcer dyspepsia. There are 10

patients in each group. H. pylori densities are evaluated using the

Sydney system, a semiquantitative score. Although there is a ten-

dency to have a higher bacterial load between the pediatric age

groups, the differences are not significant.



Discussion

Although it is generally established that PUD is a rare

pathology in pediatrics, there were no data comparing its

frequency in children and in adults. Ulcers and erosions

in the stomach and in the duodenum also occur in chil-

dren as previously shown by several studies [34–36].

PUD is, however, more frequent in older children than

in very young ones [36]. A preliminary study performed

in our center showed that PUD in H. pylori-infected

patients appears after the age of 8 years and that the

frequency of PUD gradually increases up to adulthood

(P. Bontems, S. Cadranel, unpublished data). In this

paper, we showed clearly that the frequency of PUD was

lower in children than in adults and, more interestingly,

that PUD was less frequently associated with H. pylori

infection in children than in adults.

The other question we tried to elucidate was the

possible underlying reasons explaining that ulcers in

children occur more rarely in the course of an H. pylori

infection. As the clinical outcome of the infection

depends to a large degree on the relative balance of the

immune responses, we compared innate and adaptive

mucosal response between children and adults. The

response in children is generally claimed to correspond

to an early immune response and in adults to a more

chronic response to H. pylori infection. Humoral

response was first investigated by several research

teams, who have found that this response was weaker

and that the antigens recognized in children were

different [23,24]. The Th1 type mucosal cytokine secre-

tion was also less intense [6,7,37]. The results concern-

ing the severity of gastritis were more conflicting.

Indeed, some previous studies did not show differences

in the severity of gastritis in children compared with

adults nor differences in bacterial density, atrophy, and

intestinal metaplasia being the only parameter that

showed a linear tendency to increase with age [30]. We

found the same results in a pilot study (P. Bontems,

A. Neuman-Ova, P. Heimann, J.M. Devaster, V. Segers,

C. Deprez, S. Cadranel, unpublished data). On the

contrary, Whitney et al. [31] found that in children,

the bacterial density was higher with a milder density

of neutrophils, plasma cells, and eosinophil compared

with adults. Harris et al. [25] found similar results.

Due to these inconsistent results between past stud-

ies, we decided to conduct a comparative study where

the cellular densities of immune cells were counted

manually. We found that, in the lamina propria, the

densities of neutrophils and of CD20 cells were

increased compared with the controls in all age groups.

The density of CD3 cells also increased except in chil-

dren younger than 8 years, whereas CD8 cells

increased only in adults. In the epithelium, the density

of neutrophils increased only in adults and CD3 cells

only in children of at least 8 years of age. There was no

increase in CD20 or CD8 cells. A correlation was found

between age and densities of neutrophils and of CD3

cells in the lamina propria. A correlation was also

found between age and the activation status of NF-jB.Some previous studies suggested that the recruit-

ment of neutrophils is lower or inconstant. Indeed, in

these noncomparative studies, the presence of neu-

trophils in the gastric mucosa was observed more fre-

quently than in controls but only in 35–75% of

infected children [38,39]. When comparing children

from population with high or low risk of cancer, Be-

doya et al. [40] observed that all of the components of

the inflammatory response to H. pylori infection

described in adults are present in children, but that

older children display higher degrees of neutrophil and

lymphocyte infiltrates and of H. pylori colonization than

younger ones. In our study, we only observed an

absence of neutrophils in some areas delimited by the

eyepiece graticule in children younger than 8 years (5/

10), while neutrophils were always present in older

children and in adults.

Our study has some limitations. Indeed, we mainly

studied the antral mucosa because the severity of the

gastritis using the Sydney system score always showed

more severe features in this area of the stomach. The

children were arbitrarily separated into two subgroups

using a cutoff of 8 years of age. This cutoff was decided

after a ROC curve was performed when evaluating the

accuracy of serology in children (clearly lower before

the age of 8 years) [23]. Several studies have ascribed

an increased risk of peptic ulcer and/or gastric cancer

with bacterial genes encoding virulence factors (such as

vacA genes, the cag PAI, or homB) [8,41]. In this study,

CagA-positive strains were equally distributed and

could not explain the differences observed in the densi-

ties of immune cells. However, other virulence factors

were not determined.

Interleukin-8 (IL-8) and IL-17 have been shown to

be the cytokines mainly implicated in the mucosal

recruitment of neutrophils and their secretion related

to NF-jB activation. As we were not able to measure

these cytokines, we cannot provide evidence that a

lower secretion of these cytokines due to a lower acti-

vation of NF-jB causes the failure of neutrophil recruit-

ment. One study suggested that lower neutrophil

recruitment might be due to lack of IL-8 secretion [42]

while others have shown that IL-8 protein levels were

significantly higher in the gastric mucosa of infected

children compared with uninfected ones, but the secre-

tion of IL-8 was not compared with the level observed



in adults [5,37,43–45]. Interestingly, IL-17 mRNA was

found increased in the mucosa of infected children

compared with uninfected ones [5], but IL-17 level was

found lower in infected children than in adults [27].

The activation of NF-jB in H. pylori infection was

first described by Sharma et al. [10] in vitro. Since

then, several studies have shown in vivo that the

NF-jB activity is higher in the gastric mucosa of

H. pylori-infected adults, activated NF-jB being present

in different cells in the epithelium and in the lamina

propria. NF-jB activation correlates with mucosal IL-8

secretion [46], activity of gastritis [12,13,16] (a semi-

quantitative score of neutrophil influx), and H. pylori

density [13]. In another study, however, the correlation

with bacterial density was not observed [16]. In this

study, we showed that the activation of NF-jB in

H. pylori-infected children is weaker than in adults and

correlated with neutrophil recruitment and CagA sta-

tus, but not with bacterial density. Many pathways can

be implicated in the activation of NF-jB during the

infection, and there are conflicting results to determine

the major ones in vivo [47]. Some are CagA dependent

and others CagA independent. No study so far has com-

pared the stimulation of the upstream signaling path-

ways implicated in the activation of NF-jB, such as

those mediated by TLRs and NOD-like receptors in dif-

ferent age groups. It is highly tempting to compare the

immune response of children and adults to H. pylori

infection in terms of qualitative and quantitative innate

immune signaling induced by this bacterium. One pos-

sibility is that induced inflammasome-mediated inflam-

matory pathways might be intrinsically different in

children in comparison with the adults due to the

immaturity of the innate defenses in children’s under

8 years [48]. Indeed, a recent study has shown that, in

comparison with adults, infants from birth onwards

exhibit a steady decline in IL-8 and IL-1-beta produc-

tion induced by alum, an unrelated inflammasome

activator commonly used as vaccine adjuvant [49].

In conclusion, PUD was less frequent in children

and less frequently caused by H. pylori infection. The

different clinical outcomes observed from the infection

in children, especially under the age of 8 years, can be

the consequence of the lower mucosal immune

response. We showed additional evidences that the gas-

tric mucosal recruitment of neutrophils, of CD3+, and

of CD8+ cells is lower in infected children in compari-

son with infected adults. The lower activation status of

NF-jB transcription factor in children may be either a

direct consequence of the lesser mucosal recruitment of

neutrophils, of CD3+, and of CD8+ cells or, more proba-

bly, of the existence of an overall more subtle inflam-

matory innate host defense in children.

Acknowledgements and Disclosures

We are thankful to Anouk Neuman-Ova for assistance in read-

ing the histologic sections, Nathalie Vennist for the numerous

immunohistochemical staining, and Pierre Heiman for instruct-

ing how to read sections and participation in the development

of the immunohistochemical methods. This study was

supported by grants of The Belgian Kids Foundation and the

Belgian Helicobacter study group.

Author Contributions

All authors have participated in the concept and design

of the study, interpretation of the data, drafting and

revising of the manuscript and have approved the man-

uscript as submitted. Data collection and data encoding

were performed by P Bontems and A Burette. EMSA

was performed by E Aksoy. Histologic analysis was per-

formed by P Bontems, V Segers, and C Deprez. Statisti-

cal analysis was performed by P Bontems.

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